One of the necessary components of a vaccine trial is the availability of well-characterized stocks of virus that have been titered in vitro and in vivo to determine the minimum infectious dose. Simian immunodeficiency virus (SIV)/Mne viral stocks produced in various cell lines and in peripheral blood mononuclear cells have been used in various vaccine experiments involving envelope peptides as well as recombinant vaccinia virus expressing SIV envelope proteins. The in vivo titration endpoint (animal infectious dose 50 - AID50) occurred at a 10 to the fifth-fold dilution of the viral stock. On specific clonal cell lines in vitro, this same stock can be diluted an additional two logs and shown to still contain infectious particles. A quantitative measurement of the total amount of p28 (gag CA) antigen in the same virus stocks reveals that they contain approximately 10 to the ninth virus particles per milliliter. As part of an in vivo titration study of SIV molecular and single-cell clones, macaques were inoculated intravenously or intrarectally with various dilutions of these viral stocks. The infectious dose required for a serological response after intrarectal inoculation contained approximately 10 cubed more virus than the minimum dose required for seroconversion following an intravenous inoculation. Macaques inoculated with doses of SIV below the threshold required for seroconversion and recovery of virus exhibited T-cell proliferation in response to SIV envelope synthetic peptides. Some macaques, previously exposed intravenously to subinfectious doses of SIV, were subsequently challenged 16 months later with an infectious intrarectal dose of SIV. Naive macaques (never exposed to SIV) all became infected, but previously SIV- exposed animals resisted the virus challenge. The inability to productively infect macaques previously exposed to a subinfectious dose of SIV suggests that T-cell immunity may confer long-term protection against infection, and raises the possibility that AIDS vaccines should be designed to optimize the cellular arm of the immune system.